Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP

ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [γ-32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases. Graphical overview

radioactive ATP in a reaction buffer that contains Mg 2+ . Control reactions include a sample without protein addition and an incubation of the ATPase in the presence of 1 mM orthovanadate, a classical inhibitor of P-type ATPases. In addition, a catalytically inactive mutant of the protein can serve as a negative control for the reaction and evaluate the presence of potentially contaminating ATPases present in protein preparations. The amount of non-radioactive ATP used in each reaction has to be in excess, so that it is not rate-limiting. Typically, approximately 1 mM of nonradioactive ATP is used per reaction. However, the quantity of protein, the ATP concentration, and the incubation time for the ATP hydrolysis reaction should initially be tested. For an overview, the workflow is visualized in Figure  1. The ATPase reaction is terminated by placing the samples on ice and acid addition. Subsequently, reaction components are separated by molybdate-phosphate extraction and the amount of liberated inorganic phosphate is determined via scintillation counting. Comparison to a buffer sample without protein is used for background correction. A calibration reference sample with a known volume from the [γ-32 P]-ATP stock allows calibration of the measured scintillation signal to [γ-32 P] in the sample, and thus a quantification of the hydrolyzed ATP.

Figure 1. Experimental design and workflow for measuring ATPase activity.
After preparing the samples, the ATPase mix is added, and the reaction is started by transferring the samples to the heating block. The reaction is terminated by placing the samples on ice and adding reagent A, which contains molybdate that forms a complex with free inorganic phosphate. The solution is transferred to a glass tube containing orthophosphoric acid, and reagent B is added for phosphate extraction by phase separation. After separation, 500 μL of the upper phase is added to the scintillation fluid in the scintillation vial and taken for measurement.

A. Sample preparation
We recommend preparing each sample as duplicates or triplicates. 1. Prepare test samples with a final volume of 45 μL in separate 2 mL reaction tubes on ice. An exemplary pipetting scheme is shown in Table 1. Typically, 40-250 ng of purified ATPase is used per reaction. If the specific ATPase activity range of the protein is unknown, several dilutions should be tested. Use the assay buffer to make a total of 45 μL volume. Note: It is recommended to store purified proteins at -80 °C in small aliquots to avoid multiple freezethaws. Thawing has to be done slowly on ice before an experiment is performed.

B. Preparation of the 10× ATPase reaction mix
1. To take the rate of decay of [γ-32 P]-ATP into consideration, calculate its activity for the day the assay is conducted. Manufacturers specify a calibration or reference date that corresponds to the indicated activity of a radiolabeled reagent. This date can be used to determine the residual activity of the radioactive isotope on the day the assay is conducted using the following equation:

D. Molybdate-phosphate extraction
Note: Molybdate-phosphate extraction is needed to separate inorganic 32 P-phosphate from non-hydrolyzed 32 P-ATP, as illustrated in Figure 2.

Data analysis
1. Extract the signal intensities from the .txt file and load it into Microsoft Excel for analysis. 2. Data analysis: a. First, the amount of hot ATP in the reference sample must be calculated based on its specific activity on the day of the experiment and the volume taken from the 10× ATPase reaction mix. Correlating the amount of hot ATP in the calibration reference sample with its measured cpm value allows calibration of measured cpm values. Exemplary sample values for calculation re listed in Table 3. b. Next, the sample signal can be background corrected by the buffer sample signal, and the amount of hydrolyzed hot ATP can be quantified. c. After correction for the total upper phase volume and extrapolation for hydrolyzed non-radiolabeled ATP, the total amount of hydrolyzed ATP can be specified per minute and per milligram of protein to yield the specific ATPase activity. ref. = reference sample CFVol = correction factor for total upper phase volume CFRatio = correction factor for ratio hot to cold ATP mprotein = amount of protein vii. The final specific ATPase activity can be compared between samples with and without an inhibitor.
Here, the sample without inhibitor is set to 100% to allow quantification of specific inhibition in percentage ( Figure 4).